Viral hemorrhagic fevers caused by viruses belonging to the genus Ebolavirus and Marburgvirus, both members of the Filoviridae family, are among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Ebola virus (EBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger the severe hemorrhagic syndrome. In order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Our research for this year is therefore focused on elucidating the functions of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have two ongoing projects: (1) characterization of the pathogenic processes in the Syrian hamster model that recapitulates Marburg hemorrhagic fever (MHF) and (2) characterization of EBOV protein interactions. (1) Characterization of pathogenic processes in the Syrian hamster model, which recapitulates MHF. While the NHP model is used to evaluate the efficacy of vaccines and therapeutics against filoviruses because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of filovirus research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of filoviral HFs. Recently, we have developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted EBOV that manifests many of the clinical and pathological findings observed in EBOV-infected NHPs and humans, including coagulation abnormalities. Similarly, we sought to apply our work with EBOV to establishing a rodent model for Marburg virus (MARV) strain Angola, thought to be the most virulent strain of MARV. MARV Angola was lethally adapted to both hamsters and guinea pigs through serial passage. We demonstrated that infection with hamster-adapted MARV produces severe disease in hamsters, including coagulation abnormalities evidenced by the appearance of a petechial rash that mimics that observed on humans and NHPs. Furthermore, to identify the molecular determinants of MARV virulence in rodent models, we determined the full-length genome sequences of several lethal rodent-adapted variants and identified three amino acid mutations in VP35, VP40, and VP24. Although the role that these mutations play in the acquisition of virulence during the course of rodent adaptation is unclear, based on MARV VP40s proven ability to antagonize IFN signaling and the fact that mouse-adaptation of EBOV may be linked to the ability of the virus to subvert the IFN pathway, we speculate that these three point mutations may affect the ability of MARV VP40 to inhibit IFN signaling in a species-specific manner. (2) Characterization of EBOV protein interactions. Relatively little information exists regarding the molecular details that govern interactions between EBOV proteins. As such, we are actively interested in understanding the determinants of EBOV protein interaction and the functional outcomes of those interactions. The EBOV nucleoprotein (NP) and viral protein (VP) 24, both constituents of the viral nucleocapsid, are the sole factors responsible for EBOV virulence in mice, suggesting that these two proteins play a critical role in the induction of disease. Given their contribution to EBOV virulence, we sought to characterize the physical relationship between NP and VP24. We used confocal microscopy and immunoprecipitations to demonstrate that wild-type NP both co-localizes and interacts with VP24. To determine the region on VP24 responsible for the interaction with NP, we performed bioinformatics analysis to identify the regions most likely to be involved in protein-protein interactions. Based on this prediction, we generated a series of VP24 mutants each with up to eight consecutive amino acids mutated to alanines throughout the protein. Assessing these mutants for their ability to interact with NP revealed a region near the C-terminus of VP24 that plays a critical role in the ability of VP24 to interact with NP. Further mutational analysis allowed us to resolve the interaction domain to the amino acid level. Unlike wild-type VP24, the VP24 mutants that were unable to interact with NP were also unable to support the formation of transcription/replication-competent virus-like particles (trVLPs). Interestingly, however, the ability to interact with NP was not predictive of the ability to support trVLP formation, suggesting the involvement of additional factors. The work to elucidate the physical relationship between NP and VP24 has laid the foundation for understanding the functional relationship between these two proteins. Indeed, based on this and other work, we hypothesize that VP24 plays a critical role in condensing the nucleocapsid, thereby restricting viral replication and transcription and promoting nucleocapsid packaging and egress. Future work will focus on validating this hypothesis using a variety of techniques, including trVLP systems, RNA immunoprecipitations, electron microscopy, and structural biology.